Nucleic Acids Research, 1989, Vol. 17, No. 10 3709-3723
© 1989
MOLECULAR BIOLOGY |
Characterization of early DNA synthesis in Xenopus eggs after injection of circular plasmid DNA
Department of Molecular and Experimental Medicine, Scripps Clinic and Research Foundation 10666 North Torrey Pines Road, La Jolla, CA 92037, USA
Received February 8, 1989. Revised April 18, 1989. Accepted April 18, 1989.
Circular plasmid DNA is known to replicate when injected into unfertilized eggs of Xenopus laevis. We characterized early DNA synthesis which precedes the replication. Incorporation of a radioactive precursor into covalent closed circular DNA becomes detectable as early as 2 minutes after injection. Judging from the sensitivity to aphidicolin, replicative DNA polymerase 
or 
is involved in this reaction. However, analysis of density-labeled product as well as detection of newly synthesized, unmethylated strands by restriction endonuclease Dpnl digestion, both indicated that most if not all, of the synthesized DNA is present as short repair-like patches in the injected DNA molecules. They are present randomly in all the HaeIII fragments of injected DNA, and transcriptional activation of the 5S RNA gene inserted in the plasmid does not affect the distribution. Only a minor fraction of injected DNA is utilized for this reaction, and 12% of such DNA molecules are further chased into replicated progeny DNA. This efficiency of replication is nearly the same as that of bulk injected DNA which has not been subjected to the early DNA synthesis. We concluded that, despite the common use of replicative DNA polymerase, the early DNA synthesis and the subsequent DNA replication are mutually independent processes.